Preparation of serotonine and derivatives

ABSTRACT

A process for the separation of serotonine from coffee wax wherein a solution of coffee wax is subjected to alkaline hydrolysis using a strong base in the presence of water in an inert atmosphere after which the reaction medium containing the serotonine is recovered characterized in that the solvent for the coffee wax is a compound having the general formula II: 
     
         R--(OC.sub.2 H.sub.4).sub.x --OC.sub.n H.sub.2n OH         II 
    
     wherein R is hydrogen or an alkyl group containing from 1 to 4 carbon atoms, x is 0 or 1 and n is an integer from 2 to 4 with the proviso that x cannot be 1 when n is 3 or 4. N-acetyl serotonine is prepared by acetylating serotonine to N, O-diacetyl serotonine and then selectively hydrolyzing the O-acetyl group. Melatonine is obtained by methylating N-acetyl serotonine in the 5-position. Mexamine is obtained by de-acetylating melatonine in a hot alkaline solution containing a water-insoluble alcohol and acidifying the alcohol phase with hydrochloric acid.

The present invention relates to a process for the separation ofserotonine from coffee wax and also to processes for the preparation ofcertain derivatives of serotonine, more particularly N-acetylserotonine, melatonine and mexamine.

Serotonine, the chemical name of which is 5-hydroxytryptamine, is anindolic alkaloid having the following formula: ##STR1##

This alkaloid plays an important role in the metabolism of the brain andhas vasoconstrictor, antihypertensive and antiallergenic properties, andmay be used for the treatment of psychoses, migraine and for the controlof excessive smoking.

The derivatives of serotonine also possess pharmacological properties asfollows:

N-acetyl serotonine, the chemical name of which isN-acetyl-5-hydroxytryptamine has antihypertensive properties.

Melatonine the chemical name of which is N-acetyl-5-methoxytryptamine,is secreted by the pineal gland and possesses a regulatory activity onthe circadian cycle. In addition, its use in an amount of 1-2 mg/day caninduce ovulation in sheep, which is of considerable economic importance.Moreover, it has been shown that melatonine can induce sleep in man inan amount of 1-3 mg/kg body weight.

Mexamine, the chemical name of which is 5-methoxytryptaminehydrochloride, has been proposed as a sedative and as a radioprotectiveagent.

In French Pat. Nos. 2333793 and 2371429 there is described a process forthe separation of serotonine from coffee wax in which a solution ofcoffee wax in an alcohol is subjected to an alkaline hydrolysis using astrong base in an inert atmosphere, after which the reaction mediumcontaining the serotonine is recovered. In French Pat. No. 2333793 thealcohols used are those insoluble or partially soluble in water havingfrom 4 to 8 carbon atoms e.g. isobutanol, pentanols or hexanols. Howeverthe yield obtained is relatively low (8.6 grams serotonine per kilogramof wax) because only about 50% hydrolysis takes place. In French Pat.No. 2371429 the alcohol used is benzyl alcohol or one of its homologuesbut the yield is not increased because the alcohol forms thecorresponding primary amine which is difficult to separate from theserotonine and, in addition, a β-carboline is formed from serotoninewhich impairs the yield. We have now found, surprisingly, that by usingcertain glycols or glycol ethers as the solvent in a process similar tothat described in the above-mentioned French Patents, a much morecomplete hydrolysis can be achieved without provoking the formation ofamines and β-carboline and leading to yield about 3.5 times greater.

Accordingly the present invention provides a process for the separationof serotonine from coffee wax wherein a solution of coffee wax issubjected to alkaline hydrolysis using a strong base in the presence ofwater in an inert atmosphere after which the reaction medium containingthe serotonine is recovered characterised in that the solvent for thecoffee wax is a compound having the general formula II:

    R--(OC.sub.2 H.sub.4).sub.x --O--C.sub.n H.sub.2n OH       II

wherein R is hydrogen or an alkyl group containing 1 to 4 carbon atoms,x is 0 or 1 and n is an integer from 2 to 4 with the proviso that xcannot have a value of 1 when n is 3 or 4.

The following classes of solvents are comprised within the generalformula II:

(a) ethylene glycol and its ether derivatives

(b) propylene glycols and their ether derivatives

(c) diethylene glycol and its ether derivatives

Preferred solvents are ethylene glycol monobutyl ether and diethyleneglycol monomethyl ether.

Preferably, the solvent has a boiling point of a least 120° C. and amelting point no higher than 30° C. The solvent should not substantiallyreact with the new compounds obtained nor substantially hinder the laterstages of the process and, in addition should be substantiallychemically stable under drastic conditions. The solvent enables thewater and strong base to be introduced into the lipid phase.

The amount of solvent used may be, for instance, from 20% to 75% byweight and preferably from 30% to 50% by weight based on the weight ofthe coffee wax.

As stated in the above-mentioned French Patents, serotonine is presentin coffee wax in the form of amides. The alkaline hydrolysis breaks theamide bond and liberates the serotonine as well as the correspondingfatty acids. The strong base used for the hydrolysis may be, forexample, sodium or potassium hydroxide. However, potassium hydroxide ispreferred because it does not form solid soaps with the fatty acidswhich are liberated. The various parameters which govern the alkalinehydrolysis or saponification of the coffee wax are those conventionallyused for saponification, e.g., the pH should be of the order of 12 to 14and the amount of alkali used is advantageously from 10% to 50% byweight and preferably from 20% to 40% by weight based on the weight ofcoffee wax. In addition, since the alkaline hydrolysis takes place veryslowly at lower temperatures, it is preferably carried out attemperatures from 120° C. to 160° C. and especially from 130° C. to 150°C. for a time advantageously from 3 to 6 hours. The reaction may alsotake place at temperatures from 160° C. to 180° C. for a time of from 1to 3 hours. The hydrolysis is desirably carried out at atmosphericpressure using from 3 to 10% preferably from 4 to 7.5% of water based onthe weight of coffee wax. Amounts of water greater than 10% may be usedbut cause an increase in pressure and while it is possible to useamounts above about 25% there is nothing beneficial in doing so. Thealkaline hydrolysis must be effected in an inert atmosphere, forexample, under nitrogen, because serotonine is very sensitive to oxygenin an alkaline medium. Preferably the reaction medium is agitatedvigorously.

The coffee wax that is generally available is a by-product from thedecaffeination of green coffee and therefore is rich in caffeine.Although the process according to the invention may be effected usingordinary coffee wax, it is preferred to employ decaffeinated coffee wax,because caffeine is likely to be converted into caffeidine during thealkaline hydrolysis, which lowers the yield of serotonine.

The serotonine may be isolated from the reaction medium which containsit by conventional methods, exploiting the fact that serotonine is acompound which has basic characteristics and thus has a minimumsolubility in water at pH about 10.8 (its isoelectric point).

The present invention also provides a process for the preparation ofN-acetyl serotonine characterised in that serotonine is acetylated toform N, O-diacetyl serotonine which is then treated with an alkalinemixture of water and a lower alcohol to selectively hydrolyse theO-acetyl group of the N, O-diacetyl serotonine to give N-acetylserotonine.

The serotonine used for the process may be obtained by any method, andmay conveniently be prepared from coffee wax, preferably by the processof this invention. The acetylation may be carried out by conventionalmeans, for instance, by the addition of excess acetylating agent,preferably acetic anhydride. The acetylation forms a mixture containinga major part of N, O-diacetyl serotonine together with a small amount ofthe desired N-acetyl serotonine. These two acetylated derivatives areadvantageously extracted from the acetylation medium by means of asolvent substantially insoluble in water, e.g., iso-butanol, preferablyat pH 7, and then conveniently concentrating the organic phase to obtainan oil containing the two acetylated derivatives. This oil is thenselectively hydrolysed, conveniently by dissolving in an alkalinemixture of water and alcohol in an amount from 2.5 to 7.5 times thevolume of oil, preferably at a pH above 11 to produce the N-acetylserotonine. The alcohol preferably has a boiling point below 100° C. andconveniently contains from 1 to 4 carbon atoms and is convenientlymethanol, ethanol, n-propanol or isopropanol. The selective hydrolysismay conveniently be carried out a temperature from 15° C. to 50° C.,preferably from 25° C. to 40° C., over a suitable period of time, forinstance from 15 to 60 minutes. The pH may be adjusted by the additionof 30% sodium hydroxide solution.

The present invention further provides a process for the preparation ofmelatonine characterised in that N-acetyl serotinone is methylated inthe 5-position. Any conventional methylating agent may be used,especially dimethyl sulphate which may, for instance, be added in excessto the aqueous-alcoholic solution of N-acetyl serotonine prepared ashereinbefore described. The pH is preferably above 11 and may beadjusted by adding a 30% sodium hydroxide solution while the temperaturepreferably does not exceed 45° C. During the reaction a considerablepart of the melatonine crystallises and may be separated mechanically,e.g., by filtration after which the reaction medium may be extracted, byconventional means, with a suitable solvent to recuperate the remainderof the melatonine. Examples of solvents that may be used aredichloromethane, chloroform, isobutanol and higher alcohols, ethylacetate and some fluoro-chloro alkanes e.g. Freons, Halons.

The present invention also provides a process for the production ofmexamine characterised in that melatonine is deacetylated in a hotalkaline solution containing a substantially water-insoluble alcohol andthen washed with water after which the alcohol phase is separated fromthe aqueous phase and acidified with hydrochloric acid.

Conveniently, the reaction mixture is cooled, for instance to ambienttemperature, before washing with water.

The hot alkaline solution preferably contains sodium hydroxide and thetemperature is conveniently above 90° C., and desirably at reflux. Thealcohol preferably contains from 4 to 8 carbon atoms and may be, forexample, isobutanol, a pentanol, a hexanol or a benzyl alcohol. Afterwashing with water the alcohol phase is preferably acidified to a pH ofless than 3 and then, if desired, concentrated. The mexaminecrystallises and may be separated by conventional means. The mexamine isadvantageously prepared from melatonine produced as hereinbeforedescribed in accordance with the present invention.

The following Examples further illustrate the present invention.

EXAMPLE 1 Extraction of serotonine

700 g of decaffeinated coffee wax containing 5% water were hydrolysedunder an inert atmosphere of nitrogen after the addition of 300 gethyleneglycol monobutyl ether, 200 g of potassium hydroxide and 12 g ofsodium dithionite. After 4 hours reaction at 140° C., the amides ofserotonine were completely hydrolysed, and the mixture was cooled to 75°C., diluted with 1000 g water and then acidified with 420 g of 32%hydrochloric acid. The aqueous phase which formed was separated and theorganic phase again extracted with 1200 g of 0.1% hydrochloric acid at75° C. After separation, the two aqueous phases were mixed, neutralisedto pH 7 and filtered. 3000 g of a solution containing 32 g of serotoninewere obtained.

EXAMPLE 2 Preparation of N-acetyl serotonine

To the aqueous solution of serotonine obtained in Example 1, there wereadded 40 g acetic anhydride while maintaining the pH between 8-9 with30% sodium hydroxide at 25° C. to 30° C. N, O-diacetyl serotonineformed, having the appearance of an insoluble gum, and was extractedtwice with 500 g isobutanol. The extract thus obtained was concentratedto obtain 80 g of an oil which was dissolved in a mixture containing 320parts water and 80 parts ethanol. The pH was adjusted to 12.5 with 30%sodium hydroxide and the solution maintained at about 30° C. for 30minutes, which provokes the selective hydrolysis of the O-acetyl group.The solution then contained 36 g N-acetyl serotonine.

EXAMPLE 3 Preparation of melatonine

To the aqueous alcoholic solution containing N-acetyl serotonineprepared in Example 2, there were added slowly and simultaneously 36 gof dimethyl sulphate and 20 g of 30% sodium hydroxide so that the pH wasmaintained at 12.5, while ensuring that the temperature did not exceed40° C. During this operation, a part of the melatonine formedcrystallised and this was filtered after neutralisation. Themother-liquor of crystallisation was decolourised with activated carbon,concentrated to eliminate the ethanol, then extracted withdichloromethane. After separation of the aqueous phase, the organicphase was concentrated to dryness which allowed the recuperation of afurther 28 g of crude melatonine. The two fractions, which totalled 42g, were mixed and purified by recrystallisation in a mixture containingwater and ethanol in a 75:25 ratio. 30 g of white crystals of melatoninewere obtained having a purity of 98.6%.

EXAMPLE 4 Preparation of mexamine

The melatonine obtained in Example 3 was taken up in 300 g isobutanol.To this mixture were added 30 g sodium hydroxide and 3 g of sodiumdithionite and the whole mixture was refluxed at 105° C. for 2 hoursunder nitrogen. The reaction mixture was then cooled and extracted with500 g water. The aqueous phase which contained sodium acetate formed aswell as excess sodium hydroxide, was separated. The isobutanol phase wasacidified to pH 2 with 32% hydrochloric acid and then concentrated whichinduced the crystallisation of the mexamine which was then filtered. 36g of crude mexamine was thus obtained. After recrystallisation in 96%ethanol, 30 g of white crystals of mexamine were obtained having apurity of 98.5%.

EXAMPLE 5

A similar procedure to that described in Examples 1 to 4 was followedexcept that diethyleneglycol-monomethylether was used instead ofethyleneglycol monobutyl ether as the hydrolysis solvent in theextraction of serotonine from coffee wax. 98.5% pure crystals ofmexamine were obtained.

We claim:
 1. A process for the separation of serotonine from coffee waxwherein a solution of coffee wax is subjected to alkaline hydrolysisusing a strong base in the presence of water in an inert atmosphereafter which the reaction medium containing the serotonine is recoveredcharacterised in that the solvent for the coffee wax is a compoundhaving the general formula II:

    R--(OC.sub.2 H.sub.4).sub.x --O--C.sub.n H.sub.2n OH       II

wherein R is hydrogen or an alkyl group containing from 1 to 4 carbonatoms, x is 0 or 1 and n is an integer from 2 to 4 with the proviso thatx cannot have a value of 1 when n is 3 or
 4. 2. A process according toclaim 1 characterised in that the solvent of formula II isethyleneglycol monobutyl ether or diethyleneglycol monomethyl ether. 3.A process according to claim 1 characterised in that the amount ofsolvent of formula II used is from 30% to 50% by weight based on theweight of coffee wax.
 4. A process according to claim 1 characterised inthat the hydrolysis is carried out at a temperature from 130° C. to 150°C. for a period of from 3 to 6 hours.
 5. A process according to claim 1characterised in that the hydrolysis is carried out at atmosphericpressure in the presence of from 3 to 10% by weight of water based onthe weight of coffee wax.
 6. A process according to claim 1characterised in that decaffeinated coffee wax is employed.